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A Δ11 desaturase from the oblique banded leaf roller moth Choristoneura rosaceana takes the saturated myristic acid and produces a mixture of (E)-11-tetradecenoate and (Z)-11-tetradecenoate with an excess of the Z isomer (35:65). A desaturase from the spotted fireworm moth Choristoneura parallela also operates on myristic acid substrate but produces almost pure (E)-11-tetradecenoate. The two desaturases share 92% amino acid identity and 97% amino acid similarity. There are 24 amino acids differing between these two desaturases. We constructed mutations at all of these positions to pinpoint the sites that determine the product stereochemistry. We demonstrated with a yeast functional assay that one amino acid at the cytosolic carboxyl terminus of the protein (258E) is critical for the Z activity of the C. rosaceana desaturase. Mutating the glutamic acid (E) into aspartic acid (D) transforms the C. rosaceana enzyme into a desaturase with C. parallela-like activity, whereas the reciprocal mutation of the C. parallela desaturase transformed it into an enzyme producing an intermediate 64:36 E/Z product ratio. We discuss the causal link between this amino acid change and the stereochemical properties of the desaturase and the role of desaturase mutations in pheromone evolution., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Desaturases catalyse modifications to fatty acids which are essential to homeostasis and for pheromone and defensive chemical production. All desaturases of the flour beetle Tribolium castaneum were investigated via query of the sequenced genome which yielded 15 putative acyl-Coenzyme A genes. Eleven desaturase mRNA were obtained in full length and functionally expressed in yeast. Phylogenetic analysis separated the desaturases into 4 distinct clades; one clade contained conserved beetle Δ9 desaturases, second clade was Tribolium-specific having diverse activities including Δ5, Δ9 and Δ12 desaturation and the other 2 clades had mixed insect representatives. Three members of this clade contained unusual inserted sequences of ∼20 residues in the C-terminal region and were related to desaturases that all contained similar inserts. Deletion of the entirety of the insert in the flour beetle Δ12 desaturase abolished its activity but this was partially restored by the reintroduction of two histidine residues, suggesting the histidine(s) are required for activity but the full length insert is not. Five new desaturase activities were discovered: Δ9 desaturation of C12:0-C16:0 substrates; two unprecedented Δ5 enzymes acting on C18:0 and C16:0; Δ9 activity exclusively on C16:0 and a further stearate Δ9 desaturase. qPCR analysis ruled out a role in sex pheromone synthesis for the Δ5 and Δ9/C16:0 desaturases. The flour beetle genome has underpinned an examination of all transcribed desaturases in the organism and revealed a diversity of novel and unusual activities, an improved understanding of the evolutionary relationships among insect desaturases and sequence determinants of activity., (Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.)

The Chinese tussah silkworm, Antheraea pernyi (Lepidoptera: Saturniidae) produces a rare dienoic sex pheromone composed of (E,Z)-6,11-hexadecadienal, (E,Z)-6,11-hexadecadienyl acetate and (E,Z)-4,9-tetradecadienyl acetate, and for which the biosynthetic routes are yet unresolved. By means of gland composition analyses and in vivo labeling we evidenced that pheromone biosynthesis towards the immediate dienoic gland precursor, the (E,Z)-6,11-hexadecadienoic acid, involves desaturation steps with Δ(6) and Δ(11) regioselectivity. cDNA cloning of pheromone gland desaturases and heterologous expression in yeast demonstrated that the 6,11-dienoic pheromone is generated from two biosynthetic routes implicating a Δ(6) and Δ(11) desaturase duo albeit with an inverted reaction order. The two desaturases first catalyze the formation of the (E)-6-hexadecenoic acid or (Z)-11-hexadecenoic acid, key mono-unsaturated biosynthetic intermediates. Subsequently, each enzyme is able to produce the (E,Z)-6,11-hexadecadienoic acid by accommodating its non-respective mono-unsaturated product. Besides elucidating an unusually flexible pheromone biosynthetic pathway, our data provide the first identification of a biosynthetic Δ(6) desaturase involved in insect mate communication. The occurrence of this novel Δ(6) desaturase function is consistent with an evolutionary scenario involving neo-functionalization of an ancestral desaturase belonging to a gene lineage different from the Δ(11) desaturases commonly involved in moth pheromone biosynthesis., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Sex pheromones produced by female moths of the Lasiocampidae family include conjugated 5,7-dodecadiene components with various oxygenated terminal groups. Here we describe the molecular cloning, heterologous expression and functional characterization of desaturases associated with the biosynthesis of these unusual chemicals. By homology-based PCR screening we characterized five cDNAs from the female moth pheromone gland that were related to other moth desaturases, and investigated their role in the production of the (Z)-5-dodecenol and (Z5,E7)-dodecadienol, major pheromone constituents of the pine caterpillar moth, Dendrolimus punctatus. Functional expression of two desaturase cDNAs belonging to the Delta 11-subfamily, Dpu-Delta 11(1)-APSQ and Dpu-Delta 11(2)-LPAE, showed that they catalysed the formation of unsaturated fatty acyls (UFAs) that can be chain-shortened by beta-oxidation and subsequently reduced to the alcohol components. A first (Z)-11-desaturation step is performed by Dpu-Delta 11(2)-LPAE on stearic acid that leads to (Z)-11-octadecenoic acyl, which is subsequently chain shortened to the (Z)-5-dodecenoic acyl precursor. The Dpu-Delta 11(1)-APSQ desaturase had the unusual property of producing Delta 8 mono-UFA of various chain lengths, but not when transformed yeast were grown in presence of (Z)-9-hexadecenoic acyl, in which case the biosynthetic intermediate (Z9,E11)-hexadecadienoic UFA was produced. In addition to a typical Z9 activity, a third transcript, Dpu-Delta 9-KPSE produced E9 mono-UFAs of various chain lengths. When provided with the (Z)-7-tetradecenoic acyl, it formed the (Z7,E9)-tetradecadienoic UFA, another biosynthetic intermediate that can be chain-shortened to (Z5,E7)-dodecadienoic acyl. Both Dpu-Delta 11(1)-APSQ and Dpu-Delta 9-KPSE thus exhibited desaturase activities consistent with the biosynthesis of the dienoic precursor. The combined action of three desaturases in generating a dienoic sex-pheromone component emphasizes the diversity and complexity of chemical reactions that can be catalysed by pheromone biosynthetic fatty-acyl-CoA desaturases in moths., ((c) 2010 Elsevier Ltd. All rights reserved.)

Cold-acclimated non-diapause pupae, and summer- and winter-diapause pupae of the onion maggot, Delia antiqua (Diptera: Anthomyiidae), show marked cold hardiness as compared with intact non-diapause pupae. Homeoviscous adaptation of cellular membranes is crucial to enhance the cold hardiness of organisms, and Delta9-acyl-CoA desaturases have been assumed, albeit without experimental evidence in insects, to play a key role in the adaptation. We cloned the cDNA of a desaturase gene (Dadesat) from D. antiqua, which is most likely to encode Delta9-acyl-CoA desaturase. Expression of Dadesat mRNA in the brain, midgut, and Malpighian tubules of cold-acclimated and diapause pupae was upregulated 2-10 fold, correlating well with the increase in cold hardiness. In the pupae with enhanced cold hardiness, palmitoleic and oleic acids, the presumed products of Dadesat, in the phospholipids were significantly increased. These findings suggest that the increase in the expression of Dadesat contributes to enhanced cold hardiness in D. antiqua through the production of these unsaturated fatty acids.

The pheromone blend produced by the tobacco hornworm moth (Manduca sexta) (L.) female is unusually complex and contains two conjugated dienals and trienals together with two monounsaturated alkenals. Here, we describe the identification and construction of two genes encoding MsexKPSE and MsexAPTQ desaturases from a cDNA library prepared from the total RNA of the M. sexta pheromone gland. The MsexKPSE desaturase shares a high degree of similarity with Delta(9)-desaturases from different moth species. The functional expression of MsexAPTQ desaturase in Saccharomyces cerevisiae followed by a detailed GC-MS analysis of fatty acid methyl esters (FAME) and their derivatized products and gas-phase Fourier transform infrared (FTIR) spectroscopy of the extracted FAME confirms that this enzyme is a bifunctional Z-Delta(11)-desaturase. MsexAPTQ desaturase catalyses the production of Z11-hexadecenoate (Z11-16) and Z10E12- and E10E12-hexadecadienoates (Z10E12-16) via 1,4-desaturation of the Z11-16 substrate. The stereochemistry of 1,4-desaturation and formation of isomers is discussed.

Seven desaturase cDNAs were isolated from pheromone glands of Helicoverpa assulta, a moth producing a sex pheromone blend with high Z9-16:Ald and low Z11-16:Ald, opposite to what is found in other heliothine moths such as Helicoverpa zea. Six of the seven sequences map onto recently defined lepidopteran desaturase sequence lineages and the other is orthologous to a desaturase sequence previously reported only in H. zea. The levels of desaturase-encoding transcripts in pheromone glands were determined and the three most abundant ones were functionally expressed in a desaturase-deficient mutant strain of Saccharomyces cerevisiae. The HassNPVE transcript, shown to encode a delta9 desaturase producing more Z9-18:Acid than Z9-16:Acid, was the most abundant, followed by the HassKPSE transcript, shown to encode a delta9 desaturase producing more Z9-16:Acid than Z9-18:Acid, and by the HassLPAQ transcript, shown to encode a delta11 desaturase producing only Z11-16:Acid. Thus, the relative amounts of transcripts encoding two delta9 desaturases and a single delta11 desaturase in H. assulta pheromone glands were consistent with the relative amounts of unsaturated fatty acid precursors required to produce the major and minor sex pheromone components of this species. Desaturase transcript levels in pheromone glands were also found to be as high during scotophase as during light phase, when pheromone production ceases. The other four transcripts were present at extremely low levels in H. assulta pheromone glands and the functional roles of their encoded desaturase-homologous proteins could not be determined.

The redbanded leafroller moth, Argyrotaenia velutinana (Lepidoptera: Tortricidae) uses a 92:8 mixture of (Z)-11- and (E)-11-tetradecenyl acetate in its pheromone blend. These are produced in the abdominal pheromone gland from the corresponding acids, which are biosynthesized in the gland in a 3:2 Z/E ratio by desaturation of myristoyl CoA. The delta 11 desaturase involved in this reaction exhibits unusual substrate and stereospecificities in specifically producing Z11 and E11 isomers of tetradecenoic acid, and exhibiting no activity with C16 and C18 precursor acids. This report describes the cloning and expression of the redbanded leafroller moth delta 11 desaturase, and compares its amino-acid sequence to those of other known insect Z9, Z10, Z11, and E11 desaturases. The metabolic Z9 desaturase from fat body tissue also was cloned and expressed, and found mainly to produce Z9-16:Acid and Z9-18:Acid. The open reading frame of the delta 11 desaturase encodes a protein with 329 amino acids, whereas the open reading frame of the Z9 desaturase encodes a protein with 351 amino acids. Addition of this new delta 11 desaturase with its different substrate and regiospecificites to the databank of characterized integral-membrane desaturases will be key in efforts to determine amino-acid mutations responsible for the wide array of unsaturated fatty-acid products.

The open reading frame of a gene encoding a protein homologous to vertebrate fatty acid desaturases has been characterized in Drosophila melanogaster. This is the first description of a desaturase sequence in insects. Two cDNAs were cloned: the combined 1.5 kb nucleotide sequences indicate that the gene encodes a polypeptide of 383 amino acid residues with a high sequence similarity to the well defined delta 9 desaturases from rat and yeast and desaturases from carp and tick. The Drosophila protein contains two histidine cluster motifs (HXXHH) and two hydrophobic regions which are conserved among all desaturases, whereas the amino and carboxy ends look more variable. The desaturase gene seems to be expressed in adults at a low level, with a greater prevalence in females than in males. Two genomic sequences have been amplified by polymerase chain reaction (PCR), and have a very similar open reading frame but a different organization; one with three introns and the other without introns. Both seem to correspond to two distinct genes and have been named "desat locus". They are localized to a single locus, 87C, on the right arm of the third chromosome. The possible involvement of the desat product in the biosynthesis of D. melanogaster contact pheromones is discussed.